Eduardo A. Véliz

C6 substitution of inosine using hexamethylphosphorous triamide in conjunction with carbon tetrahalide or N-halosuccinimide†

Abstract Herein we report the facile conversion of 2′,3′,5′-tri- O -acetylinosine to three different nucleoside analogs via reaction of hexamethylphosphorous triamide and an organic halide. Acetyl-protected 6-bromopurine riboside, 6-chloropurine riboside and N 6 , N 6 -dimethyladenosine can each be prepared in good yield from 2′,3′,5′-tri- O -acetylinosine, HMPT and halide. The major product of the reaction is determined by the identity of the halide used and the reaction temperature.

Probing adenosine-to-inosine editing reactions using RNA-containing nucleoside analogs.

Advances in chemical synthesis and characterization of nucleic acids allows for atom-specific modification of complex RNAs, such as present in RNA editing substrates. By preparing substrates for ADARs by chemical synthesis, it is possible to subtly alter the structure of the edited nucleotide. Evaluating the effect these changes have on the rate of enzyme-catalyzed deamination reveals features of the editing reaction and guides the design of inhibitors. We describe the synthesis of select nucleoside analog phosphoramidites and their incorporation into RNAs that mimic known editing sites by solid phase synthesis, and analyze the interaction of these synthetic RNAs with ADARs using deamination kinetics and quantitative gel mobility shift assays.

Synthesis and Evaluation of an RNA Editing Substrate Bearing 2′-Deoxy-2′-Mercaptoadenosine

The RNA-editing adenosine deaminases (ADARs) catalyze deamination of adenosine to inosine in double stranded structure found in various RNA substrates, including mRNAs. Here we describe the synthesis of a phosphoramidite of 2 ′-deoxy-2 ′-mercaptoadenosine and its incorporation into an ADAR substrate. Surprisingly, no deamination product was observed with this substrate indicating replacing the 2 ′-OH with a 2 ′-SH at the editing site is highly inhibitory. Modeling of nucleotide binding into the active site suggests the side chain of T375 of human ADAR2 to be in proximity of the 2 ′-substituent. Mutation of this residue to cysteine caused a greater that 100-fold reduction in deamination rate with the 2 ′-OH substrate.